The goal of this project is to identify key factors that regulate the genetically programmed switches in ?-like globin gene activity that occur during human development. This project employs three comparative approaches to pinpoint these regulators. First, phylogenetic footprinting takes advantage of the fact that the ?-like globin clusters of the placental mammals were derived from the same five gene cluster and that similar developmental switches in gene activity occur in all mammalian lineages. Since key components of the switching machinery are conserved, aligned ? cluster sequences from primates identify short, anciently conserved cis elements within neutral DNA. A second approach, differential phylogenetic footprinting, may reveal the cis and trans factors involved in the recruitment of simian ? genes to a fetal expression pattern. By comparing sequences from simian (fetal ?) and non-simian (embryonic ?) primates, base differences associated with this alteration in ? expression are identified and functional tests are used to test this association. Initial sequence data suggest that fine tuning of the two ? genes has proceeded along different lines among the anthropoid primates: in platyrrhines, ?2 is the major fetally expressed gene, while in catarrhines, ?l predominates. HPLC and MALDI-MS are used to characterize ?1 and ?2 in fetal and embryonic blood of several platyrrhine species. The sequence differences between platyrrhines with high ?2 and catarrhines may reveal additional elements which modulate ? gene expression; elucidation of such factors is important to the ultimate goal of manipulation of ? gene activity in individuals with defective adult globin genes.